In vitro cell culture device including cartilage and methods of using the same

ABSTRACT

The present invention relates to an in vitro cell culture device which includes a vessel comprising an inner surface, a layer of cartilage disposed on at least a portion of said inner surface, the layer of cartilage including a plurality of chondrocytes in an extracellular matrix, and a growth medium in the vessel, the layer of cartilage being bathed in the growth medium. Also disclosed is a composite cell culture prepared from the in vitro cell culture device, the composite cell culture includes a first layer including chondrocytes in an extracellular matrix, a second layer disposed on the first layer and including type I collagen, and a third layer disposed on the second layer and including cells at least partially covering the second layer. Further aspects of the present invention relate to methods of preparing an in vitro composite cell culture, methods of screening putative therapeutic agents for activity in promoting re-epithelialization of cartilaginous tissues, and methods of screening putative therapeutic agents for activity in inhibiting growth factors or proteinases.

[0001] The present invention claims the priority benefit of U.S.Provisional Patent Application Ser. No. 60/136,610, filed May 27, 1999,which is hereby incorporated by reference.

[0002] The present invention was funded by the National Institutes ofHealth, Grant No. K08-CA01659. The U.S. Government may have certainrights in the present invention.

FIELD OF THE INVENTION

[0003] The present invention relates to an in vitro cell cultureincluding cartilage, as well as various uses thereof, includingscreening for compounds which can modify cell/cell or cartilage/cellinteractions.

BACKGROUND OF THE INVENTION

[0004] Restoration of epithelial tissue after tissue injury is a complexprocess, which includes several critical events, including deposition ofextracellular matrix (“ECM”), tissue remodeling, and angiogenesis. Theseevents are coordinated with epithelial cell migration and proliferationto restore the epithelial and/or mucosal barrier (i.e., in epithelialtissues such as tracheal epithelium which secrete mucous) Thecoordination of these events is believed to involve the interactionbetween different classes of cells as well as between cells and theirextracellular matrix.

[0005] Failure of reepithelialization after injury has been observed inthe cornea (Fini et al., “Expression of Collagenolytic/GelatinolyticMetalloproteinases by Normal Cornea,” Invest. Opthalmol. Vis. Sci.31:1779-1788 (1990)) and in chronic wounds (Stacey et al., “Tissue andUrokinase Plasminogen Activators in the Environs of Venous and IschemicLeg Ulcers,” Br. J. Surg. 80:595-599 (1993); Wysocki et al., “WoundFluid from Chronic Leg Ulcers Contains Elevated Levels ofMetalloproteinases MMP-2 and MMP-9,” J. Invest. Dermatol. 101:64-68(1993); Madlener et al., “Matrix Metalloproteinases (MMPs) and theirPhysiological Inhibitors (THAPs) are Differentially Expressed DuringExcisional Skin Wound Repair,” Exp. Cell Res. 242:201-210 (1998); DiColandrea et al., “Epidermal Expression of Collagenase DelaysWound-healing in Transgenic Mice,” J. Invest. Dermatol. 11:1029-1033(1998)). Proteinases that destroy the basement membrane over whichepithelial cells migrate have been implicated as mediators in impairedcapacity to reepithelialize.

[0006] Tissue remodeling during wound healing is critical for repair ascellular migration over an appropriate ECM requires controlled andtightly regulated proteolytic degradation of the ECM, with consequentactivation or release of matrix-bound growth factors (Clark, “Basics ofCutaneous Wound Repair,” J. Dermatol. Surg. Oncol. 19:693-706 (1993);Salo et al., “Expression of Matrix Metalloproteinase-2 and -9 DuringEarly Human Wound Healing,” Lab. Invest. 70:176-182 (1994); Vaalamo etal., “Patterns of Matrix Metalloproteinase and TIMP-1 Expression inChronic and Normally Healing Human Cutaneous Wounds,” Br. J. Dermatol.135:52-59 (1996); Moses et al., “Temporal Study of the Activity ofMatrix Metalloproteinases and Their Endogenous Inhibitors During WoundHealing,” J. Cell. Biochem. 60:379-386 (1996); Martin “WoundHealing—Aiming for Perfect Skin Regeneration,” Science 276:75-81 (1997);Arumagam et al., “Temporal Activity of Plasminogen Activators and MatrixMetalloproteinases During Cutaneous Wound Repair,” Surgery 125:5887-593(1999)).

[0007] As with the above-described tissues, reepithelialization ofinjured tracheal tissues is often incomplete. The ability of respiratoryepithelial cells (“RECs”) to migrate and proliferate and restore denudedareas of the large conducting airway after injury is poor. Post-traumarestoration is pathologically manifested by the exuberant proliferationof granulation tissue and replacement of the normal respiratoryepithelium with fibroblasts (Clark, “The Commonality of Cutaneous WoundRepair and Lung Injury,” Chest. 99(Suppl.):57S-60S (1991); Grillo,“Tracheal Replacement,” Ann. Thorac. Surg. 49:864-865 (1990)). Thisoften leads to scar formation, airway stenosis, and eventual physiologiccompromise of the host respiratory tract.

[0008] There is currently no effective way to study events ofreepithelialization after injury, particularly with respect to theintraluminal events surrounding tracheal repair. Present approaches totracheal repair include resection and reanastomosing the injured airway,replacement of the damaged portion by synthetic material, and use ofautologous tissue for reconstruction of the tracheal defect (Letang etal., “Experimental Reconstruction of the Canine Trachea with a FreeRevascularized Small Bowel Graft,” Ann. Thorac. Surg. 49:955-958 (1990);Mulliken et al., Abstract, “The Limits of Tracheal Resection withPrimary Anastomosis: Further Anatomical Studies in Man,” J. Thorac.Cardiovasc. Surg. 55:418 (1968); Neville et al., “ProstheticReconstruction of the Trachea and Carina,” J. Thorac. Cardiovasc. Surg.72:525-536 (1976)). Recently, tissue engineering approaches have beentaken, including forming an in vivo tracheal cartilaginous scaffoldingby injecting dissociated chondrocytes into a preformed syntheticconstruct (Hirano et al., “Hydroxylapatite for Laryngotracheal FrameworkConstruction. Ann. Otol. Rhinol. Laryngol. 98:713-717 (1989); Okumura etal., “Experimental Study of a New Tracheal Prosthesis Made from CollagenGrafted Mesh,” Trans. Am. Soc. Artif. Organs. 37:M317-M319 (1991);Langer et al., “Tissue Engineering,” Science 260:920-926 (1993)). Suchdevices were of limited success owing to lack of reepithelialization. Inthe case of synthetic replacement, migration of the prosthesis can occurand may result in chronic ulceration, and even fatal hemorrhage (Grillo,“Tracheal Replacement,” Ann. Thorac. Surz. 49:864-865 (1990)).

[0009] A frequent problem seen in tracheal repair with synthetic orautologous materials is the failure of luminal surfacereepithelialization. Failure of reepithelialization to reestablishluminal integrity is an important reason why no acceptable surgicalprocedure exists for the repair of extended segments of tracheacompromised by inhalation injury, congenital anomalies, or neoplasticdisease.

[0010] Why the rate of reepithelialization in the large conductingairway is different from that seen within other epithelial-lined or-covered surfaces is unclear. The phenomenon of “slowed”reepithelialization is seen after both ablative surgical reconstructionand denudation injury, where the epithelium and basement membrane areremoved with an intact cartilaginous superstructure (e.g., inhalationinjury).

[0011] One of the difficulties in understanding the relationship betweenepithelium and its underlying substructure (cartilage and submucosa) isthe inaccessibility of the tissue for direct observation. It would bedesirable, therefore, to provide an in vitro cell culture which includesa developed substructure or cartilaginous layer which can be used tostudy epithelial cell development.

[0012] The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

[0013] One aspect of the present invention relates to an in vitro cellculture device which includes a vessel including an inner surface, alayer of cartilage disposed on at least a portion of the inner surface,the layer of cartilage including a plurality of chondrocytes in anextracellular matrix, and a growth medium in the vessel, the layer ofcartilage being bathed in the growth medium.

[0014] A further aspect of the present invention relates to a compositecell culture which includes a first layer including chondrocytes in anextracellular matrix, a second layer disposed on the first layer andincluding type I collagen, and a third layer disposed on the secondlayer and including cells at least partially covering the second layer.

[0015] Another aspect relates to a method for preparing an in vitrocomposite cell culture. This method is carried out by providing an invitro cartilage layer that includes chondrocytes in an extracellularmatrix, disposing a type I collagen layer on the cartilage layer, andcontacting the type I collage layer with epithelial cells underconditions effective for the epithelial cells to multiply and at leastpartially cover the layer of type I collagen.

[0016] Still another aspect of the present invention relates to a methodof screening putative therapeutic agents for activity in promotingre-epithelialization of cartilaginous tissues. According to oneapproach, the method is carried out by introducing a putativetherapeutic agent into a composite cell culture of the present inventionand then assessing epithelial cell growth on the composite cell culture,wherein increased surface area coverage of a plurality of distinctplaques of epithelial cells indicates that the putative therapeuticagent has activity in promoting reepithelialization of cartilaginoustissues. Alternatively, this method is carried out by providing an invitro cell culture device of the present invention, introducing a layerof type I collagen onto the layer of cartilage, introducing epithelialcells onto the layer of type I collagen to form a composite cellculture, introducing a putative therapeutic agent into the compositecell culture, and assessing epithelial cell growth on the composite cellculture, wherein growth and migration of epithelial cells beyonddistinct plaques thereof indicates that the putative therapeutic agenthas activity in promoting re-epithelialization of cartilaginous tissues.

[0017] Yet another aspect of the present invention related to a methodof screening putative therapeutic agents for activity in inhibiting agrowth factor or proteinase which prevents re-epithelialization ofcartilaginous tissues. According to one approach, this method is carriedout by introducing a putative therapeutic agent into a composite cellculture of the present invention and assessing epithelial cell growth onthe composite cell culture, wherein increased surface area coverage of aplurality of distinct plaques of epithelial cells indicates that theputative therapeutic agent has activity in inhibiting a growth factor orproteinase which prevents re-epithelialization. Alternatively, thismethod is carried out by providing an in vitro cell culture device ofthe present invention, introducing a layer of type I collagen onto thelayer of cartilage, introducing epithelial cells onto the layer of typeI collagen to form a composite cell culture, introducing a putativetherapeutic agent into the composite cell culture, and assessingepithelial cell growth on the composite cell culture, wherein growth andmigration of epithelial cells beyond distinct plaques thereof indicatesthat the putative therapeutic agent has activity in inhibiting a growthfactor or proteinase which prevents re-epithelialization.

[0018] The in vitro cell culture device of the present invention enablesthe growth of cells on an in vitro cartilage substructure, which enablesthe study of cell-cell interactions between chondrocytes and other celltypes introduced onto the cell culture device, as well as cell-matrixinteractions between cartilage and other cell types introduced onto thecell culture device. When collagen inserts are placed onto the cellculture device and isolated cells or tissues are introduced onto thecollagen inserts, the resulting composite cell culture can similarly beused. In addition, the in vitro cell culture device and composite cellculture can be used to screen various therapeutic agents for theirability to modify such cell-cell or cell-matrix interactions, both on acellular level as well as on a molecular level. The in vitro cellculture device and composite cell culture will facilitate thedevelopment of systems in which graft tissues can be raised in vitro forsubsequent grafting onto a patient, preferably using the patient's owncells so as to avoid any undesirable immune reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a partially exploded view of an in vitro cell culturedevice of the present invention which has been used to prepare acomposite cell culture of the present invention.

[0020] FIGS. 2A-C are enlarged images showing bovine chondrocytes inculture for 28 days after isolation.

[0021]FIG. 2A shows hematoxylin-eosin staining at 165×;

[0022]FIG. 2B shows electron microscopy at 300×; and

[0023]FIG. 2C shows type II collagen staining at 83×.

[0024] FIGS. 3A-D are enlarged images by scanning electron micrograph ofday 14 control and composite cell cultures.

[0025]FIGS. 3A and 3B illustrate the difference between confluentepithelium on the control cell culture (3A, magnification 32×) anddistinct patches of respiratory epithelium on the composite cell culture(3B, magnification 100×).

[0026]FIGS. 3C (magnification 32×) and 3D (magnification 100×)illustrate respiratory epithelium on composite cell cultures, witharrows indicating nonconfluent epithelium.

[0027]FIG. 4 is an image of a gel electrophoresis of RNA transcriptsisolated from day 14 respiratory epithelial cells from control andcomposite cell cultures. M represents an RNA ladder; lane 1, β-actinfrom control culture; lane 2, β-actin from composite culture; lane 3,transforming growth factor-β (“TGF-β from control culture; lane 4, TGF-αfrom composite culture; lane 5, transforming growth factory-β (“TGF-β”)from control culture; lane 6, TGF-β from composite culture.

[0028] FIGS. 5A-B are images illustrating the results of gelatinzymography for matrix metalloproteinase (“MMP”) activity performed onmedia conditioned by respiratory epithelial cells and chondrocytes.Pre-stained molecular weight markers and purified MMP-2 and -9 standardswere included. Serum-free media (“SFM”)and REC-conditioned media weremixed with sample buffer and run undiluted. Chondrocyte-conditionedmedia (“CCM”) was diluted 5-fold in sample buffer.

[0029]FIG. 5A represents a Coomassie blue-stained gelatin zymogram runin the absence of EDTA. Clear areas represent zones of substrate lysis.

[0030]FIG. 5B represents a zymogram incubated with the divalent cationchelator EDTA. MW, molecular weight marker proteins with individualbands indicated; lane 1, MMP standards (5 ng/n-d); lane 2, MMP standards(1 ng/ml); lane 3, SFM; lane 4, REC-conditioned medium; lane 5, day 3serum-containing CCM; lane 6, day 3 serum-free CCM. Upper arrowindicates MMP-9 (92 kDa gelatinase); lower arrow indicates MMP-2 (72 kDagelatinase).

[0031]FIG. 6 is a graph illustrating the effect of CCM in reducingproliferation of REC. Viability of the control and treated cultures wasequivalent (97.7±1.0% for control; 94.1±2.90/o for CCM). Valuesrepresent average cell number±SEM from triplicate cultures in arepresentative experiment; * indicates a significant difference (P<0.01)between control and treated cultures.

[0032] FIGS. 7A-B are images illustrating the results of gel zymographyfor MMP activity performed on chondrocyte cultured medium with either anMMP inhibitor or its negative control.

[0033]FIG. 7A shows medium collected on day 3 of culture and

[0034]FIG. 7B shows medium collected on day 7. Lane 2, MMP-2/MMP-9standards (10 ng/ml). Upper arrow indicates 92 kDa gelatinase (MMP-9)and lower arrow indicates 72 kDa gelatinase (MMP-2). Media werepre-incubated with p-arninophenylmercuric acetate to activate latentenzyme activity prior to analysis. Lane 3, SFM; lane 4, day 3 RECconditioned medium; lane 5, control SFM; lane 6, control+negative drug;lane 7, CCM; lane 8, CCM+GM600 1; lane 9, CCM+negative drug.

[0035]FIG. 8 is a graph illustrating the influence of MMP inhibitor onREC proliferation in the presence of CCM with either 10 nM GM6001, theMMP inhibitor, or control drug; similar results were obtained using 50nM. Values represent mean cell number±SEM from triplicate dishes in arepresentative experiment. Viability ranged from 91-89% and were notsignificantly different among the treatment groups; * indicates asignificant difference between control and treated groups with p<0.01and ** indicates a significant difference with p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

[0036] One aspect of the present invention relates to an in vitro cellculture device which can be used to grow cells and study cell-cartilageor cell-cell interactions, to measure the efficacy of potentialtherapeutic agents on chondrocytes or other cell types, and to growtissues with developed substructure for subsequent implantation.

[0037] The in vitro cell culture device includes a vessel having aninner surface, a layer of cartilage disposed on at least a portion ofthe inner surface, where the layer of cartilage includes a plurality ofchondrocytes in an extracellular matrix, and an amount of growth mediumin the vessel sufficient to bathe the layer of cartilage.

[0038] The vessel can be any suitable walled structure which enablesdevelopment of an in vitro cell culture device or three-dimensionalcomposite cell culture of the present invention. The vessel ispreferably formed of glass or a high-grade thermoplastic material whichis suitable for such uses.

[0039] According to one embodiment, illustrated in FIG. 1, the vessel ofthe in vitro cell culture device 10 is in the form of a petri dishhaving upper 12 and lower 14 members. The lower member 14 has an innersurface 16 defined by bottom 18 and integral sidewall 20. The layer ofcartilage 30 is present adjacent to the bottom 18. When used to grow acomposite cell culture of the present invention, as shown in FIG. 1, alayer of collagen (e.g., type I collagen) 32 is disposed on the layer ofcartilage 30, and a partial layer of cells 34 is deposited on the layerof collagen 32. As shown, the partial layer of cells 34 develops to formdiscrete patches or plaques of cells rather than a confluent layer ofsuch cells. By introducing a therapeutic agent capable of enabling cellproliferation and migration, the partial layer of cells 34 is thenallowed to grow to confluence.

[0040] The chondrocytes used in the in vitro cell culture device of thepresent invention can be any suitable type of chondrocyte. Chondrocytesare cells found in various types of cartilage, e.g., articular (orhyaline) cartilage, elastic cartilage, and fibrocartilage. Specifically,chondrocytes are mesenchymal cells that have a characteristic phenotypebased primarily on the type of extracellular matrix they produce.Precursor cells produce type I collagen, but when they become committedto the chondrocyte lineage, they synthesize type II collagen, which is asubstantial portion of the extracellular matrix. In addition, committedchondrocytes produce proteoglycan aggregate, called aggrecan, which hasglycosaminoglycans that are highly sulfated.

[0041] According to one embodiment of the present invention, thechondrocytes employed in an in vitro cell culture device are upperairway cartilage chondrocytes.

[0042] Suitable chondrocytes can be isolated from any suitable mammaliansource organism, including, without limitation, human, orangutan,monkey, chimpanzee, dog, cat, rat, mouse, horse, cow, pig, etc.

[0043] Chondrocyte cells used for preparation of the in vitro cellculture device of the present invention can be isolated by any suitablemethod. Various starting materials and methods for chondrocyte isolationare known (see generally, Freshney, Culture of Animal Cells: A Manual ofBasic Techniques, 2d ed., A. R. Liss Inc., New York, pp 137-168 (1987);Klagsburn, “Large Scale Preparation of Chondrocytes,” Methods Enzymol.58:560-564 (1979), which are hereby incorporated by reference) and areeasily reproduced by those of skill in the art.

[0044] If the starting material is a tissue in which chondrocytes areessentially the only cell type present, e.g., articular cartilage, thecells can be obtained directly by conventional collagenase digestion andtissue culture methods. Alternatively, the cells can be isolated fromother cell types present in the starting material. One known method forchondrocyte isolation includes differential adhesion to plastic tissueculture vessels. In a second method, antibodies that bind to chondrocytecell surface markers can be coated on tissue culture plates and thenused selectively to bind chondrocytes from a heterogeneous cellpopulation. In a third method, fluorescence activated cell sorting(FACS) using chondrocyte-specific antibodies is used to isolatechondrocytes. In a fourth method, chondrocytes are isolated on the basisof their buoyant density, by centrifugation through a density gradientsuch as Ficoll.

[0045] It may be desirable in certain circumstance to utilizechondrocyte stem cells rather than differentiated chondrocytes. Examplesof tissues from which stem cells for differentiation, or differentiatedcells suitable for transdifferentiation, can be isolated includeplacenta, umbilical cord, bone marrow, skin, muscle, periosteum, orperichondrium. Cells can be isolated from these tissues by explantculture and/or enzymatic digestion of surrounding matrix usingconventional methods.

[0046] Once the chondrocytes have been isolated, they are preferablyplated onto collagen inserts at a suitable cell density (i.e., about 20to about 40×10⁶ cells per well) in a suitable growth medium. A number ofsuitable chondrocyte growth media are known in the art and modificationsof known growth media can readily be made to optimize growth of suchchondrocytes and their formation of an extracellular matrix. Onesuitable growth medium includes Dulbecco's modified Eagle's medium(“DMEM”) containing 10% fetal bovine serum (FBS), 1% antibiotics and 50mu g/ml ascorbic acid (Gibco, Grand Island, N.Y.). Other known culturemedia include, without limitation, RPMI 1640, Fisher's, Iscove's orMccoy's, all of which are commercially available. Other additives mayalso be included in the chondrocyte growth medium, such asplatelet-derived growth factor (“PDGF”), which has been shown toincrease cartilage cell number without promoting further differentiationalong the endochondral differentiation pathway (see U.S. Pat. No.6,001,352 to Boyan et al., which is hereby incorporated by reference).

[0047] The collagen inserts preferably contain substantially pure type Icollagen, i.e., at least 80% type I collagen, preferably at least 85%type I collagen, and more preferably, at least 90% type I collagen. Thechondrocytes are maintained for about 21 to about 30 days or until theextracellular matrix is sufficiently developed. Suitable development ofthe extracellular matrix, denoting cartilage formation, is demonstratedby the following observations: opacity of culture, thickness of culture(i.e., between about 2-4 mm), and the firmness. This is achieved whenthe culture is brought to air interface.

[0048] Having established the development of the layer of chondrocytesin extracellular matrix within the vessel, the in vitro cell culturedevice can then be used to (i) study the relationship or interactionbetween chondrocytes and/or the extracellular matrix (“ECM”) with othercells or tissues that can be introduced into the in vitro cell culturedevice; (ii) identify putative therapeutic agents which are capable ofmodifying cell-cell or cell-ECM interactions; or (iii) grow developedtissues along with substructure for subsequent implantation.

[0049] After development of the extracellular matrix, a second layerwhich includes collagen, preferably type I collagen, is applied over thelayer of chondrocytes in the extracellular matrix (i.e., cartilage). Thetype I collagen preferably forms a substantial portion of the secondlayer. The type I collagen can readily be isolated and purified from anumber of sources or it is otherwise commercially available, forexample, from Vitrogen, Collagen Biomaterials (Palo Alto, Calif.). Thetype I collagen is preferably at least about 80% pure, more preferablyat least about 85% pure, most preferably about 90% pure. It can beapplied over the cartilage layer until a thickness of about 0.5-2 mm isachieved. Obviously, the volume of type I collagen which is needed toachieve such thickness will vary with the size of the vessel in whichthe culture resides.

[0050] On top of the second layer containing collagen is applied agrowth medium including a particular type of cell(s), whose growth inthe in vitro cell culture device is desired. Once introduced onto thesecond layer, a third layer is formed which includes the cells at leastpartially covering the second layer.

[0051] Suitable cell types which can be used to develop athree-dimensional, composite cell culture include epithelial cells,fibroblasts, endothelial cells, epidermal cells, muscle cells, orcombinations thereof. The cells can be isolated from any tissue sourceof a suitable mammalian organism. The mammalian organism can be the sameor different from the organism from which the chondrocytes wereobtained. The cells can be added to the layer of collagen at rate ofabout 1.0-10.0×10⁵ cells per cm², preferably about 1.0-5.0×10⁵ cells percm², more preferably about 1.5-3.5×10⁵ cells per cm². Suitable growthmedia include those described above, or otherwise known in the art forgrowth of particular cell types.

[0052] Once the cells have been introduced onto the collagen layer inthe in vitro cell culture device, the cells should be allowed to growuntil the cell layer has grown to confluence or until the growth of suchcells otherwise has sufficiently mimicked in vivo growth of such cells.Once the growth of cells has been established, whether to confluence ornot, the in vitro cell culture device contains a composite cell cultureof the present invention. The composite cell culture represents athree-dimensional tissue model that is particularly well adapted forstudying cell-cell or cell-substructure interactions.

[0053] A further aspect of the present invention relates to a method ofpreparing an in vitro composite cell culture. This method is carried byproviding an in vitro cartilage layer including chondrocytes in anextracellular matrix, disposing a collagen (e.g., type I collagen) layeron the cartilage layer, and then contacting the collage layer with cellsunder conditions effective for the cells to multiply and at leastpartially cover the layer of collagen.

[0054] As described above, the method can also include providing acollagen (e.g., type I collagen) substrate on which the in vitrocartilage layer can be raised. Providing the in vitro cartilage layercan be carried out by first providing a chondrocyte single cellsuspension and then culturing the chondrocytes under conditionseffective to form an extracellular matrix, thereby forming cartilage invitro. The culturing of chondrocytes, as noted above, is carried out byintroducing the chondrocyte single cell suspension onto the collagensubstrate, preferably at a cell density of about 1-10×10⁶ cells/cm².

[0055] The chondrocyte single cell suspension can be obtained accordingto any of the above-mentioned approaches for isolating chondrocytes. Apreferred approach is carried out by providing articular cartilage whichincludes chondrocytes embedded in an extracellular matrix, and thentreating the articular cartilage with collagenase II under conditionseffective to digest the extracellular matrix and produce a chondrocytesingle cell suspension.

[0056] According to one embodiment of the present invention, which isillustrated in FIG. 1, the growth of RECs in an in vitro cell culturedevice of the present invention mimics the reepithelialization ofinjured tracheal tissues. Chondrocytes are isolated from bovinearticular cartilage and cells introduced onto the collagen layer arebasal epithelial cells, secretory epithelial cells, or a combinationthereof, isolated from upper respiratory tract epithelial tissues (i.e.,bronchus, nasal polyps, or turbinates). The upper respiratory tractepithelium can be dissociated using any suitable method, for example, asdescribed in Hicks et al., Abstract, “Rapid Isolation of UpperRespiratory Cells,” Mol. Biol. Cell. 5(Suppl):118a (1994), which ishereby incorporated by reference. The isolated epithelial cells areintroduced onto the collagen layer in a suitable growth medium and at arate indicated above. The ability of RECs to migrate and proliferate torestore denuded areas of the large conducting airway after injury ispoor, often resulting in incomplete reepithelialization. This embodimentof the in vitro cell culture device mimics the in vivo pattern ofreepithelialization, yielding discrete patches of the RECs rather than aconfluent layer of RECs.

[0057] Without being bound by any particular theory, it is believed thatcommunication between epithelial cells and underlying substructure(i.e., chondrocytes and/or extracellular matrix) is responsible formodulation of epithelial cell growth and differentiation through therelease of growth factors and other proteins. Secreted agents which arebelieved to modify REC growth include matrix metalloproteinases (“MMPs”)and transforming growth factors (“TGF”).

[0058] Transforming growth factor-α (“TGF-α”) is a member of theepidermal growth factor family and plays an important role in woundhealing (Schultz et al., “Epithelial Wound Healing Enhanced byTransforming Growth Factor-α and Vaccinia Growth Factor,” Science235:350-352 (1987); Polk et al., “Increased Production of TransformingGrowth Factor-α Following Acute Gastric Injury,” Gastroenterology102:1467-1474 (1992); and Madtes et al., “Expression of TransformingGrowth Factor-α and Epidermal Growth Factor Receptor is IncreasedFollowing Bleomycin-Induced Lung Injury in Rats,” Am. J. Respir. CellMol. Biol. 11: 540-551 (1994), which are hereby incorporated byreference). Transforming growth factor β₁ (“TGF-β”) is a multifunctionalpolypeptide with differing cell-specific effects, including stimulationor inhibition of proliferation, and regulation of extracellular matrixproduction and remodeling (Massague et al., “The Transforming GrowthFactors Family,” Ann. Rev. Cell. Biol. 6:597-641 (1990); Raghow, Role ofTransforming Growth Factors in Repair and Fibrosis,” Chest.99(Suppl.):61S-65S (1991); and Santala et al., “Regulation ofIntegrin-Type Cell Adhesion Receptors by Cytokines,” J. Biol. Chem.266:23505-23509 (1991), which are hereby incorporated by reference).

[0059] MMPs constitute a family of zinc-containing proteinases acting atneutral pH, that together are capable of degrading all components of theextracellular matrix. Substrates include collagen, gelatin, elastin,fibronectin, laminin, and proteoglycans, as well as nonmatrix substratessuch as insulin-like growth factor-binding protein-3, tumor necrosisfactor-α, fibroblast growth factor receptor 1, and angiogenic factors(Sehgal et al., “Novel Regulation of Type IV Collagenase (MatrixMetalloproteinase-9 and -2) Activities by Transforming Growth Factor-β1In Human Prostate Cancer Cell Lines,” Mol. Biol. Cell. 10:407-416(1999), which is hereby incorporated by reference). They are secreted inlatent form as propeptides requiring activation for proteolyticactivity, and are inhibited by endogenous tissue inhibitors ofmetalloproteinases (Nagase et al., “Involvement of Tissue Inhibitors ofMetalloproteinases (TEMPs) During Matrix Metalloproteinase Activation,”Adv. Exp. Med. Biol. 389:23-31 (1996); Parsons et al., “MatrixMetalloproteinases,” Br. J. Surg. 84:160-166 (1997); Shapiro, “MatrixMetalloproteinase Degradation of Extracellular Matrix: BiologicalConsequences,” Curr. Opin. Cell. Biol. 10:602-608 (1999); Toi et al.,“Metalloproteinases and Tissue Inhibitors of Metalloproteinases,” BreastCancer Res. Treat. 52:113-124 (1998), which are hereby incorporated byreference). Marker protein synthesis is transcriptionally enhanced byseveral growth factors including epidermal growth factor, basicfibroblast growth factor, platelet-derived growth factor, and nervegrowth factor, and by inflammatory cytokines such as tumor necrosisfactor-α and interleukin-1. Inhibitory agents include transforminggrowth factor-P, retinoic acid, gamma interferon, glucocorticoids,progesterone, and estrogen (Nagase et al., “Involvement of TissueInhibitors of Metalloproteinases (TEMPs) During Matrix MetalloproteinaseActivation,” Adv. Exp. Med. Biol. 389:23-31 (1996); Sehgal et al.,“Novel Regulation of Type IV Collagenase (Matrix Metalloproteinase-9 and-2) Activities by Transforming Growth Factor-β1 in Human Prostate CancerCell Lines,” Mol. Biol. Cell. 10:407-416 (1999), which are herebyincorporated by reference). Inappropriate or excessive production ofMMPs may contribute to tissue destruction in arthritis, multiplesclerosis, periodontal disease, cardiovascular disease, tumorprogression, and chronic pulmonary obstructive disease (Shapiro, “MatrixMetalloproteinase Degradation of Extracellular Matrix: BiologicalConsequences,” Curr. Opin. Cell. Biol. 10:602-608 (1999), which ishereby incorporated by reference).

[0060] In particular, MMP-2 and -9 (type IV collagenases/gelatinasesproenzymes are produced by chondrocytes and play a major role incartilage remodeling and loss of extracellular matrix in osteoarthritis(Lefebvre et al., “Production of Gelatin-degrading MatrixMetalloproteinases (“Type IV Collagenases’) and Inhibitors by ArticularChondrocytes During their Dedifferentiation by Serial Subcultures andUnder Stimulation by Interleukin-1 and Tumor Necrosis Factor α,”Biochem. Biophys. Acta. 1094:8-18 (1991), which is hereby incorporatedby reference).

[0061] Since one embodiment of the in vitro composite cell culture ofthe present invention mimics the in vivo cartilage/epithelial cellinteractions following tracheal injury, this particular composite cellculture can be used to screen for putative therapeutic agents which canpromote proper reepithelialization of cartilaginous tissues. Generally,this aspect of the present invention is carried out by introducing aputative therapeutic agent into a composite cell culture or in vitrocell culture device of the present invention and assessing cell (e.g.,epithelial cell) growth on the composite cell culture.

[0062] This aspect of the present invention can be used to screenputative therapeutic agent(s) for their ability to inhibit normal invivo collagen/epithelial cell interaction or, more specifically, theeffect of matrix metalloproteinases or growth factors on such epithelialcells. Putative therapeutic agent(s) can be introduced, either alone orin combination, onto the in vitro cell culture device either before theaddition of the epithelial cells, at the same time the epithelial cellsare introduced (i.e., in the same medium), or thereafter. The putativetherapeutic agent(s) can then be screened for their ability to disruptthe undesirable inhibition of epithelial cell growth and proliferation.Statistically significant cell growth or proliferation of epithelialcells while in the presence of a putative therapeutic agent willindicate an ability of the therapeutic agent to enablere-epithelialization of cartilage substructure following trachealinjury. Effectiveness of the putative therapeutic agent can be detected,for example, by measuring increased surface area coverage of theplurality of distinct plaques. In some cases, the epithelial cells caneven grow to confluence.

[0063] According to one embodiment, the screening of putativetherapeutic agents for activity in promoting re-epithelialization ofcartilaginous tissues is carried out by introducing a putativetherapeutic agent into a composite cell culture of the present inventionand then assessing epithelial cell growth on the composite cell culture,wherein increased surface area coverage of the plurality of distinctplaques indicates that the putative therapeutic agent has activity inpromoting re-epithelialization of cartilaginous tissues.

[0064] According to another embodiment, the screening of putativetherapeutic agents for activity in promoting re-epithelialization ofcartilaginous tissues is carried out by providing an in vitro cellculture device of the present invention, introducing a layer of type Icollagen onto the layer of cartilage, introducing epithelial cells ontothe layer of type I collagen, thereby forming a composite cell culture,introducing a putative therapeutic agent into the composite cellculture, and then assessing epithelial cell growth on the composite cellculture, wherein growth and migration of epithelial cells beyonddistinct plaques thereof indicates that the putative therapeutic agenthas activity in promoting re-epithelialization of cartilaginous tissues.

[0065] According to a further embodiment, the screening of putativetherapeutic agents for activity in inhibiting a growth factor orproteinase which prevents re-epithelialization of cartilaginous tissuesis carried out by introducing a putative therapeutic agent into acomposite cell culture of the present invention and then assessingepithelial cell growth on the composite cell culture, wherein increasedsurface area coverage of the plurality of distinct plaques indicatesthat the putative therapeutic agent has activity in inhibiting a growthfactor or proteinase which prevents re-epithelialization.

[0066] According to a yet another embodiment, the screening of putativetherapeutic agents for activity in inhibiting a growth factor orproteinase which prevents re-epithelialization of cartilaginous tissuesis carried out by providing an in vitro cell culture device of thepresent invention, introducing a layer of type I collagen onto the layerof cartilage, introducing epithelial cells onto the layer of type Icollagen, thereby forming a composite cell culture, introducing aputative therapeutic agent into the composite cell culture, and thenassessing epithelial cell growth on the composite cell culture, whereingrowth and migration of epithelial cells beyond distinct plaques thereofindicates that the putative therapeutic agent has activity in inhibitinga growth factor or proteinase which prevents re-epithelialization.

[0067] Following identification of suitable therapeutic agents whichwill allow for in vitro growth of confluent cell layers in a compositecell culture of the present invention, it is possible to prepare invitro graftable tissue specimens that contain layers of cells over asubstructural layer of cartilage. Such graftable tissue specimens canthen be introduced into a patient using procedures known in the art.

EXAMPLES

[0068] The following examples are provided to illustrate embodiments ofthe present invention, but they are by no means intended to limit itsscope.

Example 1 Isolation and Culture of Chondrocytes

[0069] Chondrocytes were harvested from bovine articulator cartilageunder clean conditions, minced finely, and digested for 12 to 16 hoursat 37° C. in phosphate buffered saline containing antibiotics,collagenase II (Worthington, Freehold, N.J.), and DNAse I (Sigma-AldrichCorporation, St Louis, Mo.) as described by Klagsburn, “Large ScalePreparation of Chondrocytes,” Methods Enzymol. 58:560-564 (1979), whichis hereby incorporated by reference. Cell viability was determined bytrypan blue staining, and cell type was confirmed by staining withhematoxylineosin and antibody to extracellular type II collagen.Chondrocytes were plated on collagen inserts (Co Eagle medium starTranswell; VWR, Rochester, N.Y.) at 20 to 40×10⁶ cells per well inDulbecco DMEM/F12 (Gibco, Grand Island, N.Y.) modified with antibiotics,10% fetal calf serum, and 50-μg/mL ascorbic acid.

[0070] As shown in FIGS. 2A-B, bovine chondrocytes established inprimary culture were morphologically similar to in vivo bovinecartilage. Cartilage cultured for less than 2 months did not always formlacunae, but always produced an abundant extracellular matrix of type IIcollagen (FIG. 2C).

Example 2 Isolation of Upper Respiratory Epithelial Cells

[0071] Human tissue specimens were obtained through Manhattan Eye, Ear,Nose and Throat Hospital, New York, N.Y., under a human institutionalreview board approved protocol. Samples were healthy tissues fromsurgical procedures performed on adults. Specimens were removedaseptically, rinsed in isotonic sodium chloride solution to removedebris, and shipped at 4° C. within 24 hours of procurement. Uponreceipt of the tissue samples, upper respiratory tract epithelium frombronchus, nasal polyps, or turbinates was dissociated as described inHicks et al., Abstract, “Rapid Isolation of Upper Respiratory Cells,”Mol. Biol. Cell. 5(Suppl):118a (1994), which is hereby incorporated byreference.

Example 3 Construction of Respiratory Epithelial Cell ContainingComposite Cell Culture and Control Culture

[0072] After chondrocytes, cultured as described in Example 1, formed alayer of extracellular matrix, 0.5 mL of type I collagen (Vitrogen;Collagen Biomaterials, Palo Alto, Calif.) was added to wells, therebyforming the first two layers of the composite. After addition of thetype I collagen, upper respiratory epithelial cells harvested accordingto Example 2 were added to type I collagen-coated chondrocytes at2×10⁵/cm², in DMEM/F-12, containing antibiotics, epidermal growth factor(10 ng/mL), hydrocortisone (1 μmol/L), insulin (5 μg/mL),L-isoproterenol (1 μmol/L), and 5% fetal calf serum. Cultures were agedfor two weeks.

[0073] Upper respiratory epithelial cells harvested according to Example2 were also added at the same density onto type I collagen-coatedculture dishes (i.e., without a layer of chondrocytes in extracellularmatrix), thereby forming control cultures. The control cultures wereaged for two weeks like the composite cell cultures.

[0074] RECs grown on type I collagen formed a continuous sheet (FIGS.3A-B). In contrast, RECs grown on composite cultures did not spread toconfluence, but rather grew as discrete patches or plaques of epithelium(FIGS. 3C-D). The epithelial cell layer in both the composite culturesand on type I collagen was undifferentiated.

Example 4 Analysis of RECs and Chondrocytes for Growth Factor Expression

[0075] RECs from the aged composite cell cultures and control cultureswere removed from the cultures by microscopic dissection and processedfor RT-PCR (reverse transcriptase polymerase chain reaction. RECs werealso processed for scanning electron microscopy (model 35CF; JEOL,Japan) at 10 to 12 kV. The cartilage supernatant was taken for zymogramanalysis.

[0076] Chondrocyte cultures aged twenty-eight days were formalin fixedand processed for immunoperoxidase histochemical analysis. Rabbitpolyclonal antibody to type II collagen (DAKO, Carpinteria, Calif.) wasapplied, washed, and followed by a biotinylated universal secondaryantibody and streptavidin peroxidase (Immunon/Shandon-Lipshaw,Pittsburgh, Pa.).

[0077] In REC from day 14 composite cultures, expression of both TGF-αand TGF-β was reduced (FIG. 4, lane 4, TGF-α; lane 6, TGF-β) as comparedwith REC on type I collagen (FIG. 4, lane 3, TGF-α; lane 5, TGF-β).Relative expression of β-actin was equal (FIG. 4, lanes 1 and 2).Paraffin sections of REC grown on type I collagen and on composites wereimmunostained for TGF-α and TGF-β. Both growth factors were expressed inchondrocytes, and to a lesser extent, in epithelial cells.

[0078] Total cellular RNA was isolated from day 14 REC composite cellcultures, and REC control cultures using TriReagent (Molecular ResearchCenter, Cincinnati, Ohio) as described by the manufacturer. Under adissecting microscope, the REC layer was removed from cultures with asterile, RNAse-free spatula into TriReagent. One to five micrograms oftotal RNA was reverse transcribed using murine Moloney leukemia virusreverse transcriptase (Kawasaki, PCR Protocols: A Guide to Methods andApplications, San Diego, Calif., Academic Press (1990), which is herebyincorporated by reference). Polymerase chain reaction amplification wasperformed using primer sets for human TGF-α, TGF-β₁, and β-actin(Clontech, Palo Alto, Calif.), following manufacturer's instructions.One-tenth volumes of polymerase chain reaction products were run on 2.5%or 3% agarose gels and visualized by ethidium bromide staining. Thepolymerase chain reaction products obtained were of expected sizes(human β-actin, 838 base pairs; human TGF-β, 161 bp; and human TGF-α,297 bp).

[0079] Both TGF-α and TGF-β play crucial roles in new tissue formationand remodeling. TGF-α stimulates proliferation in cultured epithelialcells (Derynck, “Transforming Growth Factor-α: Structure and BiologicalActivities,” J. Cell Biochem. 32:293-304 (1986), which is herebyincorporated by reference), fibroblasts (DeLarco et al., “Growth factorsfrom murine sarcoma virus-transformed cells,” Proc. Natl. Acad. Sci. USA75:4001-4005 (1978), which is hereby incorporated by reference), andendothelial cells (Schreiber et al., “Transforming Growth Factor-α: AMore Potent Angiogenic Mediator than Epidermal Growth Factor,” Science232: 1250-1253 (1986), which is hereby incorporated by reference). It ischemotactic for epithelial cells in vitro (Barrandon et al., “CellMigration Is Essential for Sustained Growth of Keratinocyte Colonies:The Roles of Transforming Growth Factor-α and Epidermal Growth Factor,”Cell 50:1131-1137 (1987), which is hereby incorporated by reference) andenhances epithelial wound healing when applied topically (Schultz etal., “Epithelial Wound Healing Enhanced by Transforming Growth Factor-αand Vaccinia Growth Factor,” Science 235:350-352 (1987), which is herebyincorporated by reference). TGF-β is mitogenic for cells of mesenchymalorigin and plays a role in repair through its ability to modulateextracellular matrix formation and tissue remodeling.

[0080] When isolated human RECs were cultured on type I collagen, thecells spread to form a confluent layer as has been previously reportedby other authors (Robinson et al., “Culture of Conducting AirwayEpithelial Cells in Serum-Free Medium,” J. Tissue Cult. Method.13:95-102 (1991), which is hereby incorporated by reference). Whenplated onto composite cultures with a layer of type I collagen on top ofcartilage, the cells did not spread efficiently but formed epithelialnests. Complete reepithelialization of the surface did not occur, evenafter 3 weeks.

[0081] Due to the physical differences between REC layers on thecomposite cell cultures and control cultures, TGF-α and TGF-β expressionwas examined. Both of these growth factors were reduced in epithelialcells from 14-day composite cultures when compared with the expressionof these factors in REC cultured on type I collagen alone. This suggeststhat the cartilage modulates the behavior of epithelial cells. Withoutbeing bound by theory, it is believed that the observed diminishedexpression of TGF-α and TGF-β results from the secretion of solublefactors from the cartilage. TGF-α was expressed in cartilage, where itmay have acted on the epithelium in a paracrine manner to decrease itsexpression.

Example 5 The Role of Matrix Metalloproteinases in RespiratoryEpithelial Cell Attachment and Proliferation

[0082] Chondrocytes were harvested from bovine or porcine articularcartilage according to the procedure set forth in Example 1. Cellviability and number were detennined by Trypan blue staining andcounting using a hemocytometer. Chondrocytes were plated on collageninserts (Costar Transwell, VWR, Pittsburgh, Pa.) at 20-40×10⁶ cells/wellin DMEM/F12 containing antibiotics, 10% FBS, and 50 μg/ml ascorbic acid(prepared fresh and added every other day). After the chondrocytes hadformed a layer of extracellular matrix (approximately 2 weeks), themedium was switched to serum free (DMEM/F12 with antibiotics, 5 μg/mlinsulin, 15 μg/ml bovine pituitary extract, 10 μg/ml epidermal growthfactor, 5 μg/ml transferrin, 1 μM hydrocortisone, 10⁻⁸ M retinol, andascorbic acid). Cultures were fed from the bottom every 3-4 days. Mediumwas collected from chondrocyte cultures on days 3, 5, 7, 10, and 14after switching from 10% FBS-containing medium to SFM. The collected CCMwas cleared by centrifugation to remove cell debris, pooled at everytimepoint, and frozen at −20° C. until assayed. No protease inhibitorswere added to collected medium.

[0083] Porcine tracheal tissue obtained at necropsy was cleaned ofdebris, rinsed thoroughly with PBS, and epithelium and submucosa removedand soaked overnight at 4° C. in MEM containingantibiotics/antimycotics. Dissociated respiratory epithelial cells wereisolated using elastase digestion as described by Hicks et al. (“RapidIsolation of Upper Respiratory Epithelial Cells,” Mol. Biol. Cell.,5(Suppl.): 118a (Abstract), (1995); Hicks et al., “Isolation andCharacterization of Basal Cells from Human Upper RespiratoryEpithelium,” Exp. Cell Res. 237:357-363 (1997), which are herebyincorporated by reference) and plated on top of type I collagencoated 35mm petri dishes at 0.2×10⁶ cells/cm² in serum free medium. In selectedexperiments, cells were cultured in Biocoat type I collagen-coated 24well plates. Medium was collected for gelatin zymography as describedabove.

[0084] The proliferative effects of CCM on REC were assessed by additionof CCM diluted 1:1 with SFM. REC cultures grown on type I collagen werefed 0.1 ml CCM or control SFM daily, and media were changed completelyevery 3 days. At selected timepoints (days 5, 7, 10, and 14), cellcultures were harvested for determination of cell number by countingusing a hemocytometer. For cells cultured on type I collagen 35 mm petridishes, medium was removed and dishes rinsed twice with PBS. Type IIcollagenase at 2 mg/ml in PBS was added and the cultures incubated at37° C. with gentle shaking for 45-60 minutes to allow digestion of thecollagen substrate. Cells were collected and 10% FBS in DMEM added tostop digestion. Cells were washed once with PBS, and 2× trypsin/EDTAsolution added to further dissociate cells from the matrix and from oneanother. After incubation at 37° C. for 20 minutes, REC were againwashed with PBS/10% FBS, then resuspended in PBS and an aliquot countedusing Trypan blue dye exclusion to determine viability and cell number.For cells cultured in Biocoat 24 well plates, cells were removed bytrypsinization, washed once in PBS/FBS and re-suspended in PBS forcounting as described above. Results were similar between the twoculture methods.

[0085] In selected experiments, GM6001, a broad-spectrum matrixmetalloproteinase inhibitor, or its negative control peptide (bothavailable from Calbiochem, San Diego, Calif.), were added to cellscultured in the presence or absence of CCM.

[0086] Seven and a half percent acrylamide gels were cast with 1 mg/mlgelatin as substrate. Latent proteases in media or standards wereactivated by incubating samples with 1 mM aminophenylmercuric acetatefor 1 hour at 37° C. prior to electrophoresis.

[0087] CCM or SFM, obtained as described above, was mixed with 2× samplebuffer (20 mM DTT, 4% SDS, 50 mM Tris, 20% glycine, 0.002% Bromophenolblue), loaded without prior heating, and electrophoresed undernon-reducing conditions at 4° C. for approximately 4 hours underconstant voltage. MMP-2 and MMP-9 standards (available from Chernicon,Temecula, Calif.) and kaleidoscope prestained molecular weight standards(available from Biorad, Hercules, Calif.) were included in every gel.After electrophoresis, gels were removed and washed twice in 2.5% TritonX-100 for 15 minutes each, then incubated in developing buffer (50 mMTris-HCl, 0.2 M NaCl, 10 mM CaCl₂, 0.02% (w/v) Brij 35, pH 7.5)overnight at 37° C., with moderate shaking. Some gels were incubated indeveloping buffer containing 20 mM EDTA (ethylenediamine tetraaceticacid) to chelate Zn² and inactivate the MMPs. Gels were rinsed twicewith distilled water then stained for 1 hour in Coomassie blue stain,followed by destaining in water/methanol/acetic acid (6:3.5:0.5) untilclear areas of enzyme activity were detected against a blue background.Estimates of amounts of MMPs present in CCM were made by comparison ofknown concentrations of MMP standards with dilutions of CCM and scanningthe clear areas of substrate lysis on the gel by laser densitometry.

[0088] Experiments were performed a minimum of twice on independenttracheal REC preparations. Data were analyzed by ANOVA or t-test, asappropriate, with a level of statistical significance at P<0.05.

[0089] CCM from days 3, 5, 7, 10, and 14 was subjected to gelatinzymography on 7.5% SDS-PAGE gels. Several major bands of gelatinolyticactivity were detected (FIG. 5A). Three distinct bands were observed at105-125 kDa and two bands were detected at approximately 92 and 88 kDa.A major broad band was detected at 72 kDa with minor bands visible at65-68 kDa. Based on comparisons with purified MMP-2 and -9 standards runin parallel with the samples, the areas of enzyme activity at 92 and 88kDa corresponded to proMMP-9 and to active MMP-9. The bands of activityat 72 kDa corresponded to proMMP-2, and the lower molecular weight formsto activated forms of MMP-2. The higher molecular forms may representadditional gelatinases. When zymograms were developed in the presence ofthe chelator EDTA, enzyme activity was abolished and the clear areas oflysis disappeared, confirming the enzyme activity as belonging to matrixmetalloproteinases (FIG. 5B).

[0090] Coincident with the secretion of matrix metalloproteinase-2(MMP2, 72 kDa gelatinase) in cartilage, as determined by gel zymography,RECs that previously strongly expressed TGF-α and TGF-β had a decreasein the expression of both of these proteins in the composite cellculture.

[0091] SFM did not contain MMP activity (FIG. 5A, lane 3). Mediumconditioned by respiratory epithelial cells cultured in SFM containedbarely detectable MMP activity (FIG. 5A, lane 4). Conditioned mediumfrom chondrocytes grown for 3 days in either 10% serum-containing mediumor for 3 days after the switch to SFM exhibited considerable MMPactivity as revealed by gelatin zymography (FIG. 5A, lanes 5 and 6,respectively).

[0092] If matrix metalloproteinases were being secreted fromchondrocytes, this may be a contributing factor in failure of epithelialcells to proliferate and spread to confluence over the covering type Icollagen layer in the composite co-culture system (described in Example3, supra). To ascertain this, serum-free CCM from days 3-14 was pooledand diluted in 1:1 with serum-free medium. This medium was fed torespiratory epithelial cells plated on type I collagen, and cell numberwas determined on day 7 of culture. Cell number was reduced by 61.3% incultures that received CCM compared with controls that received SFMalone (FIG. 6), suggesting that MMPs secreted by chondrocytes mayinterfere with epithelial cell proliferation.

[0093] In order to investigate whether MMPs were directly responsiblefor diminished REC proliferation, an inhibitor of matrixmetalloproteinases (GM6001) or its control peptide were added to RECcultured with or without CCM and cell number was determined after 7 daysin culture. Addition of GM6001 or its control peptide at a concentrationof 5 nM (10 fold higher than the Kj) did not block marker proteinactivity in pooled CCM as determined by gelatin zymography (FIGS. 7A-B),nor was proliferation positively affected by addition of the inhibitor.In a second series of experiments, GM6001 or control peptide were usedat concentrations of 10 and 50 W, but once again proliferation was notrestored to control levels (FIG. 8).

[0094] Although the invention has been described in detail for thepurpose of illustration, it is understood that such detail is solely forthat purpose, and variations can be made therein by those skilled in theart without departing from the spirit and scope of the invention whichis defined by the following claims.

What is claimed:
 1. An in vitro cell culture device comprising: a vesselcomprising an inner surface; a layer of cartilage disposed on at least aportion of said inner surface, said layer of cartilage comprising aplurality of chondrocytes in an extracellular matrix; and a growthmedium in said vessel, said layer of cartilage being bathed in saidgrowth medium.
 2. The in vitro cell culture device according to claim 1,wherein said vessel is formed of glass or plastic.
 3. The in vitro cellculture device according to claim 2, wherein said vessel is in the formof a petri dish.
 4. The in vitro cell culture device according to claim1, wherein said extracellular matrix comprises type II collagen.
 5. Thein vitro cell culture device according to claim 4, wherein said type IIcollagen is a substantial portion of said extracellular matrix.
 6. Thein vitro cell culture device according to claim 1, wherein saidchondrocytes are upper airway cartilage chondrocytes.
 7. The in vitrocell culture device according to claim 1, further comprising: a secondlayer comprising type I collagen, said second layer being disposed onsaid layer of cartilage.
 8. The in vitro cell culture device accordingto claim 7, wherein said type I collagen is a substantial portion ofsaid second layer.
 9. A composite cell culture comprising: a first layercomprising chondrocytes in an extracellular matrix; a second layer,disposed on the first layer, comprising type I collagen; and a thirdlayer, disposed on the second layer, comprising cells at least partiallycovering the second layer.
 10. The composite cell culture according toclaim 9, wherein the cells are epithelial cells, fibroblasts,endothelial cells, epidermal cells, muscle cells, or combinationsthereof.
 11. The composite cell culture according to claim 10, whereinthe cells are epithelial cells.
 12. The composite cell culture accordingto claim 11, wherein the epithelial cells are upper respiratoryepithelial cells.
 13. The composite cell culture according to claim 11,wherein the epithelial cells are present as a plurality of distinctplaques dispersed throughout the third layer.
 14. The composite cellculture according to claim 13, wherein the extracellular matrix is typeII collagen.
 15. The composite cell culture according to claim 9,wherein the chondrocytes are articular or upper airway cartilagechondrocytes.
 16. The composite cell culture according to claim 15,wherein the cells are upper respiratory epithelial cells.
 17. Thecomposite cell culture according to claim 16, wherein the upperrespiratory epithelial cells are present as a plurality of distinctplaques dispersed throughout the third layer.
 18. The composite cellculture according to claim 15, wherein the extracellular matrix is typeII collagen.
 19. The composite cell culture according to claim 15,wherein chondrocytes are upper airway cartilage chondrocytes.
 20. Amethod for preparing an in vitro composite cell culture comprising:providing an in vitro cartilage layer comprising chondrocytes in anextracellular matrix; disposing a type I collagen layer on the cartilagelayer; and contacting the type I collage layer with cells underconditions effective for the cells to multiply and at least partiallycover the layer of type I collagen.
 21. The method according to claim20, wherein said providing an in vitro cartilage layer comprises:providing a chondrocyte single cell suspension; and culturing thechondrocytes under conditions effective to form an extracellular matrix,thereby forming cartilage in vitro.
 22. The method according to claim21, wherein said providing a chondrocyte single cell suspensioncomprises: providing articular cartilage comprising chondrocytesembedded in an extracellular matrix; and treating the articularcartilage with collagenase II under conditions effective to digest theextracellular matrix and produce a chondrocyte single cell suspension.23. The method according to claim 21, further comprising: providing atype I collagen substrate.
 24. The method according to claim 23, whereinsaid culturing comprises: introducing the chondrocyte single cellsuspension onto the type I collagen substrate at a cell density of about1-10×10⁶ cells/cm².
 25. The method according to claim 20, the cells arerespiratory epithelial cells.
 26. A method of screening putativetherapeutic agents for activity in promoting re-epithelialization ofcartilaginous tissues comprising: introducing a putative therapeuticagent into a composite cell culture according to claim 12 and assessingepithelial cell growth on the composite cell culture, wherein increasedsurface area coverage of the plurality of distinct plaques indicatesthat the putative therapeutic agent has activity in promotingre-epithelialization of cartilaginous tissues.
 27. A method of screeningputative therapeutic agents for activity in promotingre-epithelialization of cartilaginous tissues comprising: providing anin vitro cell culture device according to claim 1; introducing a layerof type I collagen onto the layer of cartilage; introducing epithelialcells onto the layer of type I collagen, thereby forming a compositecell culture; introducing a putative therapeutic agent into thecomposite cell culture; and assessing epithelial cell growth on thecomposite cell culture, wherein growth and migration of epithelial cellsbeyond distinct plaques thereof indicates that the putative therapeuticagent has activity in promoting re-epithelialization of cartilaginoustissues.
 28. The method according to claim 27, wherein said introducingepithelial cells and said introducing the putative therapeutic agent arecarried out simultaneously.
 29. The method according to claim 27,wherein said introducing epithelial cells is carried out after saidintroducing the putative therapeutic agent.
 30. A method of screeningputative therapeutic agents for activity in inhibiting a growth factoror proteinase which prevents re-epithelialization of cartilaginoustissues comprising: introducing a putative therapeutic agent into acomposite cell culture according to claim 12 and assessing epithelialcell growth on the composite cell culture, wherein increased surfacearea coverage of the plurality of distinct plaques indicates that theputative therapeutic agent has activity in inhibiting a growth factor orproteinase which prevents re-epithelialization.
 31. A method ofscreening putative therapeutic agents for activity in inhibiting agrowth factor or proteinase which prevents re-epithelialization ofcartilaginous tissues comprising: providing an in vitro cell culturedevice according to claim 1; introducing a layer of type I collagen ontothe layer of cartilage; introducing epithelial cells onto the layer oftype I collagen, thereby forming a composite cell culture; introducing aputative therapeutic agent into the composite cell culture; andassessing epithelial cell growth on the composite cell culture, whereingrowth and migration of epithelial cells beyond distinct plaques thereofindicates that the putative therapeutic agent has activity in inhibitinga growth factor or proteinase which prevents re-epithelialization. 32.The method according to claim 31, wherein said introducing epithelialcells and said introducing the putative therapeutic agent are carriedout simultaneously.
 33. The method according to claim 31, wherein saidintroducing epithelial cells is carried out after said introducing theputative therapeutic agent.